title: TRPV1 potentiates TGFβ-induction of corneal myofibroblast development through an oxidative stress-mediated p38-SMAD2 signaling loop.
abstract: Injuring mouse corneas with alkali causes myofibroblast expression leading to tissue opacification. However, in transient receptor potential vanilloid 1 channel (TRPV1-/-) knockout mice healing results in transparency restoration. Since TGFβ is the primary inducer of the myofibroblast phenotype, we examined the mechanism by which TRPV1 affects TGFβ-induced myofibroblast development. Experiments were performed in pig corneas and human corneal fibroblasts (HCFs). Immunohistochemical staining of α-smooth muscle actin (α-SMA) stress fibers was used to visualize myofibroblasts. Protein and phosphoprotein were determined by Western blotting. siRNA transfection silenced TRPV1 gene expression. Flow cytometry with a reactive oxygen species (ROS) reporting dye analyzed intracellular ROS. [Ca2+]I was measured by loading HCF with fura2. In organ cultured corneas, the TRPV1 antagonist capsazepine drastically reduced by 75% wound-induced myofibroblast development. In HCF cell culture, TGF-β1 elicited rapid increases in Ca2+ influx, phosphorylation of SMAD2 and MAPKs (ERK1/2, JNK1/2 and p38), ROS generation and, after 72 hrs myofibroblast development. SMAD2 and p38 activation continued for more than 16 h, whereas p-ERK1/2 and p-JNK1/2 waned within 90 min. The long-lived SMAD2 activation was dependent on activated p38 and vice versa, and it was essential to generate a > 13-fold increase in α-SMA protein and a fully developed myofibroblast phenotype. These later changes were markedly reduced by inhibition of TRPV1 or reduction of the ROS generation rate. Taken together our results indicate that in corneal derived fibroblasts, TGFβ- induced myofibroblast development is highly dependent on a positive feedback loop where p-SMAD2-induced ROS activates TRPV1, TRPV1 causes activation of p38, the latter in turn further enhances the activation of SMAD2 to establish a recurrent loop that greatly extends the residency of the activated state of SMAD2 that drives myofibroblast development.
authors.affiliation: Department of Biological Sciences, State University of New York, State College of Optometry, New York, New York, United States of America.
authors.forename: Yuanquan
authors.initials: Y
authors.lastname: Yang
authors.forename: Zheng
authors.initials: Z
authors.lastname: Wang
authors.forename: Hua
authors.initials: H
authors.lastname: Yang
authors.forename: Lingyan
authors.initials: L
authors.lastname: Wang
authors.forename: Stephanie R
authors.initials: SR
authors.lastname: Gillespie
authors.forename: J Mario
authors.initials: JM
authors.lastname: Wolosin
authors.forename: Audrey M
authors.initials: AM
authors.lastname: Bernstein
authors.forename: Peter S
authors.initials: PS
authors.lastname: Reinach
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000468:Alkalies; D000818:Animals; D002118:Calcium / Q000378:metabolism; D003315:Cornea / Q000378:metabolism*; D065306:Corneal Injuries; D003318:Corneal Opacity / Q000139:chemically induced / Q000235:genetics* / Q000378:metabolism; D025461:Feedback, Physiological; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism; D048055:Mitogen-Activated Protein Kinase 8 / Q000235:genetics / Q000378:metabolism; D048056:Mitogen-Activated Protein Kinase 9 / Q000235:genetics / Q000378:metabolism; D058628:Myofibroblasts / Q000378:metabolism* / Q000473:pathology; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D051899:Smad2 Protein / Q000235:genetics* / Q000378:metabolism; D013552:Swine; D050916:TRPV Cation Channels / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D046509:Tissue Culture Techniques; D016212:Transforming Growth Factor beta / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics* / Q000378:metabolism
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; D000468:Alkalies; D017382:Reactive Oxygen Species; C497782:SMAD2 protein, human; D051899:Smad2 Protein; D050916:TRPV Cation Channels; C494482:TRPV1 protein, human; D016212:Transforming Growth Factor beta; D048056:Mitogen-Activated Protein Kinase 9; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048055:Mitogen-Activated Protein Kinase 8; D048051:p38 Mitogen-Activated Protein Kinases; D002118:Calcium